Microbial community analysis of the hypersaline water of the Dead Sea using high‐throughput amplicon sequencing

Abstract Amplicon sequencing using next‐generation technology (bTEFAP ®) has been utilized in describing the diversity of Dead Sea microbiota. The investigated area is a well‐known salt lake in the western part of Jordan found in the lowest geographical location in the world (more than 420 m below sea level) and characterized by extreme salinity (approximately, 34%) in addition to other extreme conditions (low pH, unique ionic composition different from sea water). DNA was extracted from Dead Sea water. A total of 314,310 small subunit RNA (SSU rRNA) sequences were parsed, and 288,452 sequences were then clustered. For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon–Wiener index curve plot reached a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. Archaea was found to be dominating the sequences (52%), whereas Bacteria constitute 45% of the sequences. Altogether, prokaryotic sequences (which constitute 97% of all sequences) were found to predominate. The findings expand on previous studies by using high‐throughput amplicon sequencing to describe the microbial community in an environment which in recent years has been shown to hide some interesting diversity.


| INTRODUCTION
The Dead Sea is a well-known salt lake located in the western part of Jordan. This natural lake represents a unique habitat for extremophiles due to its extreme geophysicochemical characteristics. For instance, the Dead Sea surface is considered as the Earth's lowest elevation on land (more than 420 m below sea level) (Avriel et al., 2011). With respect to its salinity, the Dead Sea is characterized by extreme salinity, about 34%, which is approximately 10 times higher than the salinity of oceans (Bodaker et al., 2010). Moreover, the salinity in the Dead Sea is expected to increase in the upcoming years due to high evaporation and low regional precipitation. Former research articles have already reported that the negative water budget leads to a 1-mol/L annual decrease in the Dead Sea level (Bodaker et al., 2010). In the Dead Sea water, magnesium is the dominant cation and the pH is low (around 6) (Oren, 2007). In addition, the Dead Sea area was found to have a unique UV radiation since UVA/UVB ratio is greater at the Dead Sea than anyplace else in the world (Avriel et al., 2011). Altogether, the geophysicochemical characteristics exemplify extreme conditions for living organisms and likely represent a unique microbial ecology.
In the last 5 years, several studies on samples taken from Dead Sea water, soil, and black mud were carried out to isolate and characterize the microorganisms flourishing in the Dead Sea area and to explore their biological activity (Jacob, 2012;Jacob & Irshaid, 2012Jacob, Wink, & Wink, 2016). In these studies, the majority of isolates from the Dead Sea water were bacteria (Jacob, 2012). The isolated bacteria were members of both Gram-positive bacteria (like Arthrobacter sp., Kocuria erythromyxa, and Bacillus licheniformis) and Gram-negative bacteria (like Salinivibrio costicola, Vibrio alginolyticus, and Chromohalobacter salexigens). More recently, 13 morphologically different isolates of halophilic Actinobacteria from the soil and the black mud of the Dead Sea were isolated (Jacob et al., 2016). The isolates were found to be species of the genera Brevibacterium, Rhodococcus, Leifsonia, Arthrobacter, Propionibacterium, Corynebacterium, and Trueperella. Some isolates have a pronounced antimicrobial activity (Jacob et al., 2016).
These previous studies have utilized culture-dependent methods to isolate and characterize the microorganisms in the Dead Sea.
However, we believe that most of the microbial species are still unseen in this unusual area due to the limitations of the methods used, that is, the culture-independent methods. Even though culture-dependent methods are advantageous over culture-independent, if a microbial material is required for further studies, the major disadvantage of culture-dependent methods is that they are thought to undervalue the microbial number and composition of the studied samples (Al-Awadhi et al., 2013).
In this study, we aim to explore the diversity of microbial species in the Dead Sea water using culture-independent approach. Similar studies were conducted in the last years with certain types of samples from the Dead Sea. For instance, Rhodes, Oren, and House (2012) have investigated the 16S rRNA amplicon libraries from a Dead Sea haloarchaeal bloom in 1992 and compared it with the 2007 residual population and simulated blooms in experimental microcosms. Important population shifts were detected during the bloom, and unexpectedly a signature from the bloom was retained 15 years later. In another study, Bodaker et al. (2010) and Rhodes et al. (2012) analyzed the 16S rRNA genes from particulate matter obtained from a depth of 5 m at an offshore station via tangential filtration. Their results showed that members of the family Halobacteriaceae are existent in the water column.
Analysis of 16S rRNA genes indicated that the Dead Sea, despite the harsh conditions to life, supports a unique and diverse community of halophilic Archaea (Bodaker et al., 2010;Rhodes et al., 2012).
In this current study, we explore the microbial communities in the Dead Sea water during June 2015 by metagenomic analysis. We aim in this study to determine which types of halophilic and/or halotolerant microbial taxa are present in the Dead Sea water by amplicon sequencing using the novel next-generation technology (bTEFAP ® ).
We also aim to determine the abundance of the detected taxa and the sequencing depth was evaluated to determine if it is sufficient to capture the full scope of microbial diversity. This study describes the new uncultivated species found in the Dead Sea water and represents a source data for future studies since this environment is changing continuously.

| Water sampling
Water samples (1 L) were collected in June 2015 from the surface water of the Dead Sea (31°36.545 N, 35°36.648 E). Geographic coordinates and elevation were determined using eTrex Legend C (GARMIN, Taiwan). Samples were collected in sterile clean glass bottles and transported directly to the laboratory for further processing and analysis.

| Physicochemical analysis water
The in situ temperature and pH were determined by using the portable pH/temperature meter (Model TI 9000, WalkLAB, Trans Instruments, USA). Salinity was measured by a handheld salinity refractometer. The concentrations of Na, K, Ca, and Mg were determined using flame atomic absorption spectrometry (FAAS) (Varian Spectra AA, Australia). Air-acetylene flame was used. For each element, the measurements were made using its specific hollow cath-

| DNA extraction
Water samples were filtered through 0.2 μm membranes under vacuum. The membranes were then cut outv into small pieces.
Pieces were then transferred to a 50 mL centrifuge tube. DNA extraction was then completed using E.Z.N.A. ® Water DNA kit (Omega Biotech Ltd., India) as per the manufacturer's instructions.
The eluted DNA was stored at −20°C for further analysis.

| Alpha diversity
The number of OTUs at the species level was apprised to define alpha diversity among the different groups. Alpha diversity essentially evaluates how many unique bacterial species are within the sample.

| Physicochemical analysis
The in situ temperature of the Dead Sea water sample was 22°C and the pH was 5.9. Salinity was found to be extremely high (34%). The concentration of Na, K, Ca, and Mg were also determined. Table 1 shows the concentration of the aforementioned elements in the Dead Sea water sample.

| Metagenomic analysis
After strict quality sequence curation, a total of 314,310 sequences were parsed and 288,452 were then clustered. Prokaryotic sequences (which constitute 97% of all sequences) were found to predominate and the sequences of Archaea were found to be more abundant than those of Bacteria (52% of the detected sequences belong to Archaea, whereas 45% belong to Bacteria; Figure 1). Minor abundance of eukaryotes was also detected (3%) (Figure 1).
After analyzing the Archaeal sequences, 100% of the sequences were found to belong to the phylum Euryarchaeota in which the family Halobacteriaceae is the dominant (Figure 2). The dominant genus in Halobacteriaceae was found to be Halorhabdus (52%) followed by the genus Natronomonas with 16% abundance. Other genera of Archaea were also detected, but with low abundance like Haloplanus (4%), For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon-Wiener index curve plot ( Figure 5) reaches a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity.

| DISCUSSION
As revealed by the physicochemical analysis, the Dead Sea water is characterized by high salinity (34%), and therefore it was described as a typical example of a thalassohaline brine (Oren, 2007). Additionally, the pH was found to be relatively low (pH 5.9) indicating an acidic condition in water. The high salinity and acidic pH represent extreme conditions for living organisms. Moreover, some major cations were also determined in the Dead Sea water, namely Na, K, Mg, and Ca.
The major cation that is present in highest concentration was found to be Mg, followed by Na, Ca, and K. This sequence of abundance agrees with the findings of other researchers who found that Mg, not Na, is the dominant cation in the Dead Sea water (Ma'or et al., 2006;Oren, 2007).
A metagenomic analysis of Dead Sea water was carried out by amplicon sequencing using next-generation technology (bTEFAP ® ) to explore the diversity of microbial communities in the Dead Sea.  (Häusler, 2014). This conclusion agrees with our results that showed that Proteobacteria and Firmicutes are the two major groups of Bacteria in the Dead Sea (both constitute 98% of bacterial sequences).
In this current study, we have also detected the minor abundance of eukaryotes in the Dead Sea water (only 3%). However, it should be noted that the primers used in this study (515F and 806R) are aiming at Bacteria and Archaea. Therefore, any eukaryotic sequence obtained is due to reaction with eukaryotic plastids, mitochondria, or chloroplast. Therefore, the eukaryotic sequence data were removed and were not considered.
F I G U R E 4 The main Bacterial genera in Dead Sea water and their relative abundance. Two main genera predominate: Acinetobacter (45% abundance) and Bacillus (35% abundance). Other genera were detected but with low relative abundance (3% or less) F I G U R E 5 Shannon-Wiener index (a) and rarefaction curve (b). Shannon-Wiener index and rarefaction curve were calculated based on 97% sequence similarity This study highlights the ecological diversity that can be encountered in the Dead Sea as natural brine and serves to expand on previous studies that did not detect the significant bacterial community members of the Dead Sea. Because the Dead Sea is changing over time, additional studies to evaluate regional and seasonal changes in microbial populations of the Dead Sea are needed. Moreover, biomining of isolates are underway and should expand on the initial finding presented within.